Soluble adenylyl cyclase (SACY) is an essential component of cAMP-signalling cascades that activate sperm motility and capacitate sperm. SACY activity is stimulated by HCO3– and Ca2+. Sperm from Sacy–/– (null) mice were immotile or weakly motile, but cAMP analogues N6,2′-O-dibutyryladenosine 3′,5′-cyclic monophosphate (dbcAMP) and adenosine 3′,5′-cyclic monophosphate acetoxymethyl ester (cAMP-AM) activated motility. Null sperm activated by dbcAMP quickly developed hairpin bends at the junction of the midpiece and principal piece, which could be prevented by omitting HCO3–. Treating Sacy–/– sperm with thimerosal or NH4Cl to raise flagellar cytoplasmic Ca2+ could not substitute for cAMP analogues in activating motility; however, sperm activated with cAMP-AM hyperactivated after thimerosal treatment. Treating activated wild-type sperm with SACY inhibitor KH7 did not prevent hyperactivation from developing during capacitation in vitro, although high doses impaired motility. These results indicate that, while the SACY/cAMP signalling pathway is required for motility activation, it is not directly involved in triggering hyperactivation.